Quality control of carbohydrates, especially polysaccharides and oligosaccharides, in herbal materials remains a challenge due to their complicated structures and macro-molecular mass. Generally, isolation and purification followed by complete structural characterization, namely homogeneity and molecular weight determination, compositional monosaccharide analysis, glucosidic linkage type confirmation and then repetitive structural unit speculation, etc., is the most reliable method for quality evaluation of polysaccharides and oligosaccharides in herbal materials. As known in the art, however, the relevant methodologies are extremely intricate, difficult and time consuming and therefore not suitable for routine quality control method development. Besides, two kinds of analytical methods, total sugar content determination as well as sugar composition analysis, have been widely employing for quality control of carbohydrates in herbal materials. But the methods are still defective. Total sugar content determination by colorimetric method bears very poor specificity and is therefore inapplicable for qualitative purpose. And sugar composition analysis needs complicated operations, like acid hydrolysis, derivation, and followed by qualitative and quantitative determination of sugar derivatives using HPLC (high performance liquid chromatography) or GC (gas chromatography) (FIG. 1A). Hence, the experimental results are always affected by multiple factors in the tedious operating procedure, e.g. the temperature, reaction time and acid concentration of acid hydrolysis, and are therefore significantly variable. Furthermore, the method might be one-sided and could not reflect the original existence of polysaccharides and oligosaccharides before acid hydrolysis. Consequently, more convenience and reasonable method is imperative for quality control of carbohydrates in herbal materials.
Dendrobium is one of the largest genera in the plant family Orchidaceae. More than 1100 species of Dendrobium have been identified, with a wide distribution throughout Asia, Europe and Australia. The fresh or dried stems of about thirty Dendrobium species are collectively regarded as a famous tonic herb, namely Shihu in Chinese. Documented as a “superior grade” herb in “Shennong Bencao Jing”, an ancient textbook on herbal materials in China, Dendrobium has been used for thousands of years for its traditional nourishing properties, such as benefiting stomachs, supplementing body fluids and strengthening immunity. Among them, Dendrobii Officinalis Caulis, called Tiepi Shihu in Chinese, which is derived from dried stems of Dendrobium officinale Kimura et Migo, is traditionally recognized as the best Shihu for tonic purpose, such as nourishing stomach, protecting throat and benefiting eyes. In “Dao Zang”, a classic encyclopedia of Taoism, Dendrobii Officinalis Caulis was ever listed as the first one of “the Nine Herbs for Immortality of China”. Nowadays, due to extremely scarce wild resource and distinguished tonic effects, it has been the most renowned and rarest Dendrobium herbs and has become one of the most expensive herbs in herbal markets worldwide, particularly in Southeast Asia. The stems of D. officinale are always heated and then twisted to a spiral or spring form followed by drying for sell in herbal markets and commonly known as Tiepi Fengdou (FIG. 2). The uncharacteristic appearance and high price of Tiepi Fengdou could lead to the occurrence of the adulterants, confused species, and counterfeits. Authentication and quality evaluation of Tiepi Fengdou is therefore crucial for ensuring the safety and efficacy.
Continuous efforts have been made for quality control of D. officinale based on qualitative and/or quantitative characterization, but the methods used are far from satisfactory. This herb has a unique chemical profile, in which carbohydrates account for up to 70%, along with some small molecules, such as bibenzyls and phenanthrenes, etc. Quality evaluation focusing on small molecules failed to efficiently distinguish D. officinale from other Dendrobium species. And in these studies, the investigated constituents were less than 0.21% of the whole herb material. In other words, more than 99% components in these samples were uncontrollable by these methods. On the other hand, with dominant content and proved bioactivities, carbohydrates are naturally the target in quality control of D. officinale. Nevertheless, as mentioned above, quality control of carbohydrates in D. officinale, just like other saccharide-dominant herbal materials, is also confronted with methodological bottleneck.
In Chinese Patent No. CN102370891A an approach focuses on small molecules and uses GPC column and UV detector, which is a complex and complicated approach not suitable for quick, efficient and low-cost application, is disclosed. The disclosed invention is hard to scale for commercial scale processing. While in another Chinese Patent No. CN101716283A and the publication in Journal of Pharmaceutical and Biomedical Analysis entitled Comparison of polysaccharides from different Dendrobium using saccharide mapping by J. Xu et. al, both disclosures are targeted on large molecule polysaccharides, which are also the target molecules of the present invention. However, the chemical markers they used are not the original Dendrobium polysaccharides, but the products of enzyme hydrolysis of the original Dendrobium polysaccharides. These documents disclosed a fingerprint of oligosaccharides produced by enzyme hydrolysis of Dendrobium polysaccharides, which would require complicated and time-consuming protocols, including water extraction, ethanol precipitation, deproteination, enzyme hydrolysis, and electrophoresis or HPLC.
Thus, there still exists a need for a quality control and authentication method of D. officinale that: 1) is rapid and low-cost; 2) with a mechanism that is easy-to-understand; 3) is simple; 4) is repeatable and reproducible in a satisfactory manner; 5) is practicable for both qualitative and quantitative analysis; 6) is reliable with large number of sample batches, and 7) is practical for commercial application.
Citation or identification of any reference in this section or any other sections of this application shall not be construed as an admission that such reference is available as prior art for the present application.